Adler, C.

Blaskovics, M.

Curtius, H.-C

Giudici, T. A.

Viscontini, M.

Wetzel, E.

RERO DOC Digital Library

Adler et ai: 7-substituted pterins 25 Pteridines Voi. 3, pp. 2 5 - 2 8 Short Communication 7-Substituted Pterins: Loading Experiments with 3'-Mono Deuterated Tetrahydro-L-Biopterin C. Adler, H.-C. Curtius", E. Wetzel, T. A. Giudici*, M. Blaskovics** and M. Viscontini Universitätskinderspital Zürich, Abteilung Klinische Chemie, Steinwiesenstr. 75, CH-8032 Zürich, Switzerland * Division of Genetics, Childrens Hospital, Los Angeles, U. S. A. ** Department of Pediatrics, Kaiser Permanente, Fontana, U.S.A. (Received March 1992) Introduction Since 7-substituted pterins have been detected in the urine of patients with an atypical, transient form of hyperphenylalaninemia the origin of these metabolites has been unclear for a long time. The following pterins have been found so far: the 7-isomer of L-biopterin referred to as L-primapterin, the 7-isomer of D-neo-pterin referred to as D- or L-anapterin, and the 6-oxo-7-isomer of 7-oxo-L-biopterin referred to as 6-oxo-L- or D-primapterin (1). Two findings make it plausible that these compounds derive from their 6-analogues: on the one hand, orally loading of a primapterinuric patient with L-tetrahy-drobiopterin (L-BH4) led to an increase of both L-biopterin and L-primapterin (2). And, in vitro incu-bation of L-tetrahydrobiopterin with pterin-4a-car-binolamine dehydratase-free phenylalanine hydro-xylase results in the formation of L-primapterin (3, 4). Analogue in vitro incubation experiments with D-tetrahydroneopterin also led to the formation of the 7-substituted isomer. For in vivo investigations of L-primapterin biosyn-thesis we loaded a primapterinuric patient with side-chain mono-deuterium labelled L-BH4 ([3'-2H]]-L-BH4) (5). If L-primapterin originates from L-BH4, deuterium labelling should also be present in L-pri-mapterin isolated from the urine after loading, and it can also be determined whether the side-chain is from the same origin. " Author to whom correspondence should be addressed. Material and Methods Loading of a 5 year old male primapterinuric patient T. S. was performed with a dose of 10 mg deuterated L-BH4 per kilogram body weight. His urine was col-lected up to 12 hours after loading. The purification and isolation of urinary L-biopterin and L-primapterin after loading was performed using several different chromatographic and filtration steps: After acidic oxidation of the urine with iodine the acidic solution (0.1 Ν HCl) was applied to a Dowex 1 x 4 column. The column was washed with 0.1 Ν HCl and fractions fluorescencing blue were collected. Purines were separated from pterins by precipitation in aqueous ammonia (pH 8 — 9). The alkaline solution was applied to a Dowex 50 χ 8 column where pterins and salts passed through. The collected blue fluores-cent fractions were made alkaline (pH 8 — 9) and then applied on a Dowex 1 x 4 column. Pterins were eluted with 0.1 Ν HCOOH. The eluate was made alkaline with NH 4 OH and applied to a Dowex 1 χ 4 column and the pterins were fractionated with a 0.1 Ν N H 4 + C O C r buffer, pH 7.8. The pterin solution was made alkaline with N H 4 O H (pH 8 — 9) and ap-plied to a second Dowex 1 x 4 column. The column was washed with water, and the pterins were eluted with 0.1 Ν HCOOH. Excess acid was removed by evaporation, and the residue was diluted with water. The separation of the pterins was performed using a preparative reverse-phase HPLC column (6). After the first preparative HPLC-separation L-primapterin fractions still contained some L-biopterin. Therefore a second preparative HPLC was necessary. Pteridines / Vol.3 / No. 1/2 Copyright © 1992 Walter de Gruyter • Berlin • New York 26 Adler et al.: 7-substituted pterins 100.Θ-1 5 0 . 0 -117 H j i ^ O T M S CH. 147 iL· Μ "Ε 10Θ 100.0-1 108 133 124 11Θ 120 " Τ * · 139 139 143 171 158 163 173 148 150 16Θ 170 182 139 180 190 198 200 117 5 0 . 0 -147 Μ " 110 133 125 ' ' Ι · ' Τ * * 130 140 Τ * 171 f=8 164 179 187 ??3 197 ι •' • • 11 11-ε 100  120 130 140 150 160 170 180 190 200 Figure 1. Low molecular fragments of L-primapterin. a) reference compound; b) isolated from urine after L-[3'-2H,]BH4-loading. Adler et al.: 7-substituted pterins 27 100.0-1 5 0 . 0 -100.0-, 5 0 . 0 -510 481 523 Μ.Έ 450 4Ç0 470 488 490 500 510 520 Figure 2. High molecular fragments of L-primapterin. a) reference compound; b) isolated from urine after L-[3'-2H,]BH4-loading. 28 Adler et al.: 7-substituted pterins Using the above described method of purification and isolation 300 μ§ of L-biopterin and 50 μg of L-pri-mapterin were isolated from 200 ml urine. Gas-chromatography/mass-spectrometry was per-formed using the TMS-derivatives of L-biopterin and L-primapterin (7). Results and Discussion We synthesized 3'-mono deuterated L-biopterin and analyzed it by GC/MS as a TMS-derivative. These data were compared with the MS data of unlabelled L-biopterin. Three ions were affected by a + 1 Da shift, i.e. m/z 117 containing the C-2',3'-end of the side-chain, m/z 510 originating from M + -15 often observed for TMS-derivatives and the molecular ion at m/z 525. L-biopterin as well as L-primapterin have the same electron impact-GC/MS fragmentation pattern, and differ only in their retention times on the GC column. In the GC/MS spectrum of L-biopterin, isolated after the loading, a shift of + 1 Da was observed for the primary ions expected to exhibit deuterium incorpo-ration, i. e. m/z 117, 510, and 525, were shifted to m/z 118, 511, and 526, respectively. Ion intensity values for m/z 118, 511, and 526 were found to have in-creased by 40% for this deuterium labelled sample relative to the standard (data not shown). The same is true for isolated L-primapterin after loading with deuterated L-biopterin. Again, all three relevant ions (m/z 117, 510, and 525) are shifted + 1 Da (absolute intensity increase ~ 40%) indica-tive of deuterium incorporation (see Fig. 1 and 2). This indicates clearly that the isolated L-primapterin originates from L-biopterin and, in addition, that the side-chain of L-primapterin derives from L-biopterin. With this experiment we can exclude an intermolec-ular reaction where the side-chain attaches to an al-ready existing pterin ring nucleus. Acknowledgement This project is financially supported by the Swiss National Science Foundation, project no. 31-28797.90. References 1. Curtius, H.-Ch., Küster, T., Matasovic, Α., Blau, Ν. & Dhondt, J.-L. (1988) Biochem. Biophys. Res. Commun. 153. 715-721 . 2. Curtius, H.-Ch., Matasovic, Α., Schoedon, G., Küster, T., Guibaud, P., Giudici, T. & Blau, Ν. (1990) J. Biol. Chem. 265, 3923-3930. 3. Curtius, H.-Ch., Adler, C., Rebrin, I., Heizmann, C. & Ghisla. S. (1990) Biochem. Biophys. Res. Commun. 172, 1 0 6 0 - 1 0 6 6 . 4. Davis, M. D., Kaufman, S. & Milstien, S. (1991) Proc. Natl. Acad. Sci. 88, 385-389. 5. Adler, C„ Curtius, H.-Ch., S. & Viscontiní, M. (1990) Helv. Chim. Acta 73, 1058-1063. 6. Niederwieser, Α., Staudenmann, W. & Wetzel, E. (1982) in: Biochemical and Clinical Aspects of Pteridines (Wächter, H., Curtius, H.-Ch. & Pfleiderer, W„ eds.) pp. 81 -102 , Walter de Gruyter, Berlin. New York. 7. Küster, T. and Niederwieser, A. (1983) J. Chromatogr. 278, 245-254. Pteridines / Vol. 3 / No. 1/2 

7-Substituted Pterins: Loading Experiments with 3'-Mono Deuterated Tetrahydro-L-Biopterin

Abstract

Similar works

Full text

Available Versions

RERO DOC Digital Library